I am currently designing my CRISPR protospacers and choosing my PAM sites so that I can knock out a gene from a fungal organism. I found the sequence of my gene of interest from FungiDB (NCBI database) and I found the shotgun sequencing contig containing my gene of interest.
The issue is that the gene sequence in the genomic shotgun sequencing contig is in the reverse orientation. My expected start site is on the antisense strand and the sequence is reversed (GTA). I am unsure about how to proceed and what this reverse orientation means. Was the genomic sequence somehow accidentally flipped/reversed? Should I try to reverse compliment the genomic sequence? In its current state, the gene would need to be transcribed from the lagging strand. Should I design protospacers complimentary to the lagging strand rather than the leading strand?
Any information/advice is greatly appreciated. I have hit a wall with this problem since the direction of the gene will determine where my protospacers go and which PAM site I choose.
Mitchell, I think you should take a step back and draw out the double-stranded target on a piece of paper, mark the 5' and 3' ends of each strand. Next draw a zoomed in version of your CRIPSR targets showing their specific sequences (and, again, with 5' and 3' ends marked). Then draw out the double-stranded sequence of your DNA construct + promoter (5'/3' marked), and the corresponding transcript (5'/3' marked). Then align the guide region in the transcript sequence to the genomic target. If it aligns in the proper orientation, you are good to go. At present, it is clear that you don't fully grasp DNA structure and the processes of transcription and replication ("lagging strand" is not what you think it means). If you draw out the molecules you are planning to design, hopefully, everything should make sense.
Hi Mitchell,
When you design your guide RNA, the simple principle is that you should make sure that it is identical to the targeting genomic sequence in base pairs and orientation (doesn't really matter whether it is the + or the - strand if you generate DSB as the CAS9 doesn't really care). For the flanking sequences for your construct, again, as Mark alluded to, you need to make sure that the sequences that you have are identical to their corresponding genomic sequences in base pairs AND orientation.
One more thing. If you want to KO the gene by deleting it and/or replacing it with a dominant marker, it is better to design two guide RNAs targeting regions close to the 5' and 3' ends of the ORF, respectively, and use them together. This way, you will generate two DSBs flanking the majority of the ORF, and have a better chance that the piece in the middle will be lost.
Hope it helps and good luck.
Best,
Sheng